Exon skipping is a promising strategy for Duchenne muscular dystrophy (DMD) disease-modifying therapy. To make this approach safe, ensuring that excluding one or more exons will restore the reading frame and that the resulting protein will retain critical functions of the full-length dystrophin protein is necessary. However, in vivo testing of the consequences of skipping exons that encode the N-terminal actin-binding domain (ABD) has been confounded by the absence of a relevant animal model. We created a mouse model of the disease recapitulating a novel human mutation, a large de novo deletion of exons 8-34 of the DMD gene, found in a Russian DMD patient. This mutation was achieved by deleting exons 8-34 of the X-linked mouse Dmd gene using CRISPR/Cas9 genome editing, which led to a reading frame shift and the absence of functional dystrophin production. Male mice carrying this deletion display several important signs of muscular dystrophy, including a gradual age-dependent decrease in muscle strength, increased creatine kinase, muscle fibrosis and central nucleation. The degrees of these changes are comparable to those observed in mdx mice, a standard laboratory model of DMD. This new model of DMD will be useful for validating therapies based on skipping exons that encode the N-terminal ABD and for improving our understanding of the role of the N-terminal domain and central rod domain in the biological function of dystrophin. Simultaneous skipping of exons 6 and 7 should restore the gene reading frame and lead to the production of a protein that might retain functionality despite the partial deletion of the ABD.

中文翻译：

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CRISPR / Cas9生成的Duchenne肌营养不良症的小鼠模型概括了人类DMD基因中新发现的430 kb大缺失。

跳过外显子是杜兴氏肌营养不良（DMD）疾病缓解疗法的一种有前途的策略。为了使该方法安全，必须确保排除一个或多个外显子将恢复阅读框架，并确保所得蛋白质将保留全长肌营养不良蛋白蛋白质的关键功能。但是，由于缺乏相关的动物模型，对编码N端肌动蛋白结合域（ABD）的外显子跳过后果的体内测试已感到困惑。我们创建了该疾病的小鼠模型，概述了一种新的人类突变，即在俄罗斯DMD患者中发现的DMD基因外显子8-34的从头大量缺失。此突变是通过删除X连锁小鼠D md的外显子8-34实现的该基因使用CRISPR / Cas9基因组编辑，导致阅读框移位和功能性肌营养不良蛋白的产生。携带这种缺失的雄性小鼠显示出肌肉营养不良的几个重要征兆，包括肌肉强度逐渐下降，肌酸激酶增加，肌肉纤维化和中央成核。这些变化的程度与mdx中观察到的程度相当小鼠，DMD的标准实验室模型。DMD的这种新模型将有助于基于跳过编码N末端ABD的外显子的疗法验证疗法，并有助于增进我们对N末端结构域和中央杆结构域在肌营养不良蛋白生物学功能中的作用的了解。同时跳过第6外显子和第7外显子应可恢复基因阅读框并导致产生蛋白，尽管ABD部分缺失，该蛋白仍可保留功能。